The research is to determine the effects of chemical stimuli on the biosynthesis and metabolism of (R)-NMSal (a Parkinsonian neurotoxin) and to characterize the cellular uptake and release of D-Ser (a recently identified neurotransmitter /modulator) under ischemic conditions. To achieve the research goals, new chiral analytical methods based on microchip electrophoresis-tandem mass spectrometry (MCE-MS/MS) will be developed for high throughput chiral analysis of single cells. We plan to covalently attach chiral selector molecules onto shortened single walled carbon nanotubes and then to immobilize the chiral selector-bonded carbon nanotubes in the channel, producing highly effective and stable chiral MCE separation channels. A new microchip design that enables a direct and facile coupling of MCE with a nano-ESI assembly of a mass spectrometer is also proposed and will be assessed. After the chiral MCE-MS/MS method is in place, the proposed metabolic studies will be carried out. Although it is well documented that (R)-NMSal induces Parkinsonism in rats, study on its biosynthesis and metabolism is far from adequate. We plan to incubate PC-12 or SH-SY5Y cells with deuterium-labeled salsolinol (i.e. Sal-,,,1-d4) or (R)-NMSal. After incubation, both extracellular and intracellular levels of the compounds of interest will be quantified by using the developed chiral MCE-MS/MS method. We expect that more metabolites will be detected from single cell analysis because the intracellular concentrations of metabolites are much higher than their extracellular concentrations. Therefore, a more accurate metabolite profile will be obtained, leading to a better understanding of the biosynthesis and metabolism of this neurotoxin. We also aim to investigate the cellular uptake and release of D-Ser under ischemic conditions. Some lab evidences indicated that D-Ser was involved in causing ischemic brain damage. However, no studies on the responses of nerve cells to ischemia in terms of processing and utilizing D-Ser have been carried out so far. We will deploy PC-12 cells and cultured cortical neurons exposed to oxygen-glucose deprivation (OGD) as in vitro ischemia models in this research. The cells will be incubated with either D-Ser-2,3,3-d3 (D-Ser-d3) or L-Ser- 2,3,3,-d3 under normal or OGD conditions. Both intracellular and extracellular D-Ser-d3 will be quantified by analyzing the culture medium or through single cell analysis at different time points (1, 5, 10, 30, 60, 120 min). In parallel, OGD insult-induced cell injury will be assessed by Caspase-3 assay. For the above stated three specific aims, our working hypotheses are: 1) highly efficient and durable chiral MCE separation channels can be prepared by immobilizing chiral selector-bonded carbon nanotubes in the channel, which will lead to the development of chiral MCE-MS/MS methods for high throughput chiral analysis of single cells; 2) exposure to alcohol or oxidative stress- inducing Mn2+ affects the biosynthesis and metabolism of (R)-NMSal; 3) cellular uptake and release of D-Ser is altered under ischemic conditions as a result of the cells' responses to ischemia. The chiral MCE-MS/MS analytical methods developed in this project will have long-term value for biomedical research, particularly for probing cellular metabolism involving chirality. The metabolic studies on (R)-NMSal and D-Ser will contribute to our understanding of certain neurological diseases at the molecular biology level including the neurological significance of D-Ser under ischemic conditions and the mechanism by which (R)-NMSal induces Parkinsonism. Key words: Novel bioanalytical methods, chiral microchip electrophoresis-mass spectrometry, metabolic study at cellular levels, Parkinsonian neurotoxin, (R)-N- methylsalsolinol, D-serine, ischemia. PUBLIC HEALTH RELEVANCE: The research proposed in this SC1 application aims to determine the effects of chemical stimuli on the biosynthesis and metabolism of (R)-NMSal (a Parkinsonian neurotoxin) and to characterize the cellular uptake and release of D-Ser (a recently identified neurotransmitter /modulator) under ischemic conditions. To achieve the research goals, new chiral analytical methods based on microchip electrophoresis- tandem mass spectrometry (MCE-MS/MS) will be developed for high throughput chiral analysis of single cells. Successful development of the proposed chiral MCE-MS/MS methods will have long-term value for biomedical research, particularly for probing cellular metabolism involving chirality. The metabolic studies on (R)-NMSal and D-Ser will contribute to our understanding of certain neurological diseases at the molecular biology level including the neurological significance of D-Ser under ischemic conditions and the mechanism by which (R)-NMSal induces Parkinsonism.